New Insights on Plasmin Long Term Stability and the Mechanism of Its Activity Inhibition Analyzed by Quartz Crystal Microbalance

We used the research quartz crystal microbalance (RQCM) to monitor regulatory effects of plasmin and trypsin in the presence of their inhibitor α2-antiplasmin. The gold surface of quartz crystals was modified with a β-casein layer that served as a substrate for protease digestion. The addition of plasmin or trypsin as well as their mixtures with α2-antiplasmin resulted in an increase of resonant frequency, f, and in a decrease of motional resistance, Rm, depending on the molar ratio of protease: antiplasmin. At equimolar concentrations of protease and α2-antiplasmin (5 nM:5 nM) full inhibition of protease activity took place. Monitoring of plasmin activity on an hourly and daily basis revealed a prominent effect of autolysis and decrease of plasmin activity in freshly activated samples. The degree of inhibition as well as plasmin half-life (t1/2 = 2.48 ± 0.28 days) connected with its degradation was determined.

Towards comprehensive plasma proteomics by orthogonal protease digestion

Rapid and consistent protein identification across large clinical cohorts is an important goal for clinical proteomics. With the development of data-independent technologies (DIA/SWATH-MS), it is now possible to analyze hundreds of samples with great reproducibility and quantitative accuracy. However, this technology benefits from empirically derived spectral libraries that define the detectable set of peptides and proteins. Here we apply a simple and accessible tip-based workflow for the generation of spectral libraries to provide a comprehensive overview on the plasma proteome in individuals with and without active tuberculosis (TB). To boost protein coverage, we utilized non-conventional proteases such as GluC and AspN together with the gold standard trypsin, identifying more than 30,000 peptides mapping to 3,309 proteins. Application of this library to quantify plasma proteome differences in TB infection recovered more than 400 proteins in 50 minutes of MS-acquisition, including diagnostic Mycobacterium tuberculosis (Mtb) proteins that have previously been detectable primarily by antibody-based assays and intracellular proteins not previously described to be in plasma.

Development of an Interdigitated Electrode-Based Disposable Enzyme Sensor Strip for Glycated Albumin Measurement

Glycated albumin (GA) is an important glycemic control marker for diabetes mellitus. This study aimed to develop a highly sensitive disposable enzyme sensor strip for GA measurement by using an interdigitated electrode (IDE) as an electrode platform. The superior characteristics of IDE were demonstrated using one microelectrode of the IDE pair as the working electrode (WE) and the other as the counter electrode, and by measuring ferrocyanide/ferricyanide redox couple. The oxidation current was immediately reached at the steady state when the oxidation potential was applied to the WE. Then, an IDE enzyme sensor strip for GA measurement was prepared. The measurement of fructosyl lysine, the protease digestion product of GA, exhibited a high, steady current immediately after potential application, revealing the highly reproducible measurement. The sensitivity (2.8 nA µM−1) and the limit of detection (1.2 µM) obtained with IDE enzyme sensor strip were superior compared with our previously reported sensor using screen printed electrode. Two GA samples, 15 or 30% GA, corresponding to healthy and diabetic levels, respectively, were measured after protease digestion with high resolution. This study demonstrated that the application of an IDE will realize the development of highly sensitive disposable-type amperometric enzyme sensors with high reproducibility.

Role of misfolded tau in the onset and progression of brain toxicity after trauma

ABSTRACTTraumatic brain injury (TBI) is associated with widespread tau pathology in about one third of patients. We previously found that TBI induces a transmissible tau pathology (tauTBI), with late cognitive decline and synaptic dysfunction. To understand whether tauTBI is a marker of ongoing neurodegeneration or a driver of functional decline, we employed C. elegans. Brain homogenates from chronic TBI mice, or from mice in which tauTBI had been transmitted by intracerebral inoculation, impaired C. elegans motility and neuromuscular synaptic transmission. Brain homogenates from tau P301L transgenic mice, or pre-aggregated recombinant tau, induced a similar toxic response. Protease digestion or pre-incubation of homogenates with anti-tau antibodies abolished toxicity, and TBI brain homogenates from tau knock-out mice had no toxic effect. These results support a vital role of abnormal tau species in chronic neurodegeneration after TBI and set the groundwork for the development of a C. elegans-based platform for screening anti-tau compounds.

Enzyme Mimetic Activity of ZnO-Pd Nanosheets Synthesized via a Green Route

Recent developments in the area of nanotechnology have focused on the development of nanomaterials with catalytic activities. The enzyme mimics, nanozymes, work efficiently in extreme pH and temperature conditions, and exhibit resistance to protease digestion, in contrast to enzymes. We developed an environment-friendly, cost-effective, and facile biological method for the synthesis of ZnO-Pd nanosheets. This is the first biosynthesis of ZnO-Pd nanosheets. The synthesized nanosheets were characterized by UV–visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy, transmission electron microscopy, and energy-dispersive X-ray. The d-spacing (inter-atomic spacing) of the palladium nanoparticles in the ZnO sheets was found to be 0.22 nm, which corresponds to the (111) plane. The XRD pattern revealed that the 2θ values of 21.8°, 33.3°, 47.7°, and 56.2° corresponded with the crystal planes of (100), (002), (112), and (201), respectively. The nanosheets were validated to possess peroxidase mimetic activity, which oxidized the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in the presence of H2O2. After 20 min of incubation time, the colorless TMB substrate oxidized into a dark-blue-colored one and a strong peak was observed at 650 nm. The initial velocities of Pd-ZnO-catalyzed TMB oxidation by H2O2 were analyzed by Michaelis–Menten and Lineweaver–Burk plots, resulting in 64 × 10−6 M, 8.72 × 10−9 Msec−1, and 8.72 × 10−4 sec−1 of KM, Vmax, and kcat, respectively.